There is a great demand for methods to detect and quantify the presence of a target nucleic acid sequence of interest. In particular, there is a great demand for methods that can be used to detect and quantitate the presence of a target nucleic acid in vivo and in vitro.
For example, detection of RNA in vivo is of particular interest. During the last several years, knowledge on multiple functional roles of RNAs in gene expression has substantially increased, as has the number of newly isolated RNA molecules. Additionally, it has become clear that mRNA localization plays an important role in directing specific proteins to their correct position within the cell, thus regulating gene expression and cell development (Johnston, 1995; Oleynikov & Singer, 2003). To fully understand functions of various RNAs, methods of studying their dynamic behavior within living cells are required.
Different strategies for labeling RNA molecules for in vivo detection have been used (for a review, see Pederson, 2001). Most methods rely on different RNA-specific hybridization probes and microinjection or lipofection for probe delivery to the nucleus or cytoplasm. For example self-ligating quenching probes were successfully used by Kool and co-workers for RNA hybridization within bacterial cells (Sando & Kool, 2002). Fluorescent 2-O-methyl-RNA probes have been shown to be more stable than unmethylated oligonucleotides and have been used by several groups (Carma-Fonseca et al, 1999; Molenaar et al., 2001). Molecular beacons, (Sokol et al., 1998; Perlette & Tan, 2001), RNA hybridizing oligonucleotide probes capable of fluorescence resonance energy transfer (FRET) (Matsuo, 1998; Tsui et al., 2000; Tsuji et al, 2001; Sei-Iida et al., 2000) and caged-fluorescein labeled antisense oligonucleotides present different variants of the same approach (for a review see Politz, 1999, Pederson, 2001).
However, all of these approaches are probe-based, and thus are limited by the low sensitivity of hybridization due to the low concentration of RNA within the cell. In most cases, only highly abundant RNA species can be detected. Thus, because of their abundance, it has been possible to detect β-actin mRNA, c-fos mRNA, basic fibroblast growth factor RNA, and total poly (A)-RNA).
Another obstacle of using oligonucleotide probes for RNA detection in vivo is their fast accumulation in the nucleus (Tsuji et al., 2000; Molenaar et al., 2001). One approach to overcome this difficulty has been the use short oligonucleotide hybridization probes bound to streptavidin via biotin-straptavidin interactions, to lower their passage through nuclear pores (Tsuji et al., 2000).
Yet another strategy to study RNA in vivo has been to generate fusions between functionally important RNA-binding proteins and fluorescent proteins, which allows RNA kinetic studies. For example, real time RNA movement in yeast cells was monitored by co-expressing a GFP-MS2 fusion protein and an ASH1 RNA reporter fused to MS2 binding sequences (Bertrand et al, 1998; Beach et al., 1999; Oleynikov & Singer, 2003). In these studies, GFP or its spectral variants were used as fluorescent tags. Thus, no signal amplification was generated in this approach, which again restricts the method to detection of only highly abundant RNAs. Another drawback of this approach is the background signal of the expressed fluorophore-protein chimera, which substantially limits the sensitivity of the method.
The majority of detection methods have relied on the use of nucleic acid probes to detect a nucleic acid of interest. Probe-based assays are useful in the detection, quantitation and analysis of nucleic acids. Nucleic acid probes have long been used to analyze samples for the presence of nucleic acid from bacteria, fungi, virus or other organisms and are also useful in examining genetically-based disease states or clinical conditions of interest. Nonetheless, probe-based assays have been hampered in part by difficulties associated with specificity, sensitivity and reliability, and the challenges presented by detection of nucleic acids in vivo.
Nucleic acid hybridization is a fundamental process in molecular biology. Sequence differences as subtle as a single base (point mutation) in very short oligomers (<10 base pairs “bp”) can be sufficient to enable the discrimination of the hybridization to complementary nucleic acid target sequences as compared with non-target sequences. However, nucleic acid probes of greater than 10 bp in length are generally required to obtain the sequence diversity necessary to correctly identify a unique organism, disease state or clinical condition of interest.
Several nucleic acid probe-based methods have been developed for the detection of nucleic acids. Most are designed around the amplification of selected targets and/or probes composed of DNA, including the polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), amplification with Q.beta. replicase (Birkenmeyer and Mushahwar, J. Virological Methods, 35:117-126 (1991); Landegren, Trends Genetics, 9:199-202 (1993)), rolling circle amplification (RCA), and linear rolling circle amplification (LRCA), which uses a primer annealed to the circular target DNA molecule before adding DNA polymerase.
However, these methods are associated with a number of difficulties, including relatively low precision in quantitative measurements, being laborious, expensive, time-consuming, inefficient, and lacking in sensitivity. In addition, there are sources of error in such methods, such as where structural differences lead to different efficiencies.
Any hybridization of a nucleic acid probe to a closely related non-target sequence will result in the generation of undesired background signal. Because the sequences are so closely related, point mutations are some of the most difficult of all nucleic acid modifications to detect using a probe-based assay. Numerous diseases, such as sickle cell anemia and cystic fibrosis, are caused by a single point mutation of genomic nucleic acid.
Furthermore, all of these methods suffer from a lack of sensitivity, especially to rare genetic events, such as infrequent mutations.
In addition, the detection of nucleic acids in vivo, including in real time, has presented a significant challenge. For example, demonstrating hybridization between an antisense oligonucleotide and its mRNA target has proven extremely difficult in living cells. The development of molecular beacon technology has provided one new approach to nucleic acid detection in vivo. See Sokol et al., Proc. Natl. Acad. Sci. USA 95:11538-43 (1998); Perlette et al., Anal. Chem. 73:657A (2001).
Accordingly, it would be highly desirable to have improved methods for the detection of target nucleic acids, including methods for in vivo detection. Such improved detection of DNA and RNA targets would be useful in the detection, analysis and quantitation of nucleic acid containing samples.